The invention relates to the use of at least folic acid or a folate and tetrahydrobiopterin (BH4) or derivatives thereof for treating or preventing cardiovascular or neurological disorders by modulation of the activity of nitric oxide synthase (NOS). The present invention also relates to the use of at least folic acid or a folate and tetrahydrobiopterin (BH4) or derivatives thereof for the production of a pharmaceutical preparation suitable for influencing the nitric oxide (NO) level, particularly by modulation of the activity of nitric oxide synthase (NOS) by reducing superoxide (O2) production and enhancing nitric oxide (NO) synthesis. This effect occurs in the absence of any negative changes in other risk factors, e.g. lipids, blood pressure and homocysteine. Clinical areas of application include all anomalies of the nitric oxide level, particularly the prevention and treatment of cardiovascular and of neurological disorders. The present invention also relates to pharmaceutical preparations comprising at least one compound selected from the group consisting of 5-formyl-(6 S)-tetrahydrofolic acid, 5-methyl-(6 S)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolic acid, 5,10-methanyl-(6R)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid, 5-formimino-(6 S)-tetrahydrofolic acid or (6 S)-tetrahydrofolic acid, together with tetrahydrobiopterin (BH4) or pharmaceutically compatible salts thereof and with pharmaceutically compatible active and adjuvant substances, such as arginine for influencing the nitric oxide (NO) level.
Within this text the term a folate or a derivative thereof, if not explicitly defined otherwise, always refers to the natural and unnatural stereoisomeric form of each substance, pharmaceutically compatible salts thereof and any mixtures of the isomers and the salts. As drugs, tetrahydrofolates have predominantly been used hitherto as the calcium salt of 5-formyl-5,6,7,8-tetrahydrofolic acid (leucovorin) or of 5-methyl-5,6,7,8-tetrahydrofolic acid (MTHF) for the treatment of megaloblastic folic acid deficiency anemia, as an antidote for increasing the compatibility of folio acid antagonists, particularly of aminopterin and methotrexate in cancer chemotherapy (xe2x80x9cantifolate rescuexe2x80x9d), for increasing the therapeutic effect of fluorinated pyrimidines and for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis, for increasing the compatibility of certain antiparasitic agents, for instance trimethoprim-sulfamethoxazole, and for decreasing the toxicity of dideazatetra-hydrofolates in chemotherapy and for influencing the homocysteine level, particularly for assisting the remethylation of homocysteine.
The term tetrahydrobiopterin (BH4) or a derivative thereof, if not explicitly defined otherwise, always refers to all natural and unnatural stereoisomeric forms of tetrahydrobiopterin, pharmaceutically compatible salts thereof and any mixtures of the isomers and the salts. The term tetrahydrobiopterin also includes any precursors of tetrahydrobiopterin, especially 7,8-dihydrobiopterin. (6R)-tetrahydrobiopterin is a naturally occuring cofactor of the aromatic amino acid hydroxylases and is involved in the synthesis of the three common aromatic amino acids tyrosine, phenylalanine, tryptophan and the neurotransmitters dopamine and serotonin. It is also essential for nitric oxide synthase catalysed oxidation of L-arginine to L-citrullin and nitric oxide. Tetrahydrobiopterin is involved in many other biochemical functions, many of which have been just recently discovered.
The term arginine, if not explicitly defined otherwise, always refers to the natural and unnatural stereoisomeric form of arginine. L-arginine, a natural amino acid, is the precursor of endogenous nitric oxide (NO), which is a ubiquitous and potent vasodilator acting via the intracellular second-messenger cGMP. In healthy humans, L-arginine induces peripheral vasodilation and inhibits platelet aggregation due to an increased NO production. Both an excess and a lack of production of NO have been linked to pathological conditions, including cardiovascular disorders, septic shock, inflammation and infection, and brain damage in stroke and neurological disorders. The term nitric oxide synthase (NOS), if not explicitly defined otherwise, always refers to all isoforms endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS).
Nitric oxide (NO) has been identified as a mediator of atherosclerosis. Therefore it is a therapeutic target in cardiovascular prevention trials. It also plays an important role in neurological disorders. Biological effects of nitric oxide (NO) are not limited to vascular relaxation, but are also important in the respiratory, urogenital and gastrointestinal system, central and peripheral nervous system, neuroendocrine and endocrine systems, and nonspecific immunity.
Nitric oxide (NO) and superoxide (O2.) are cytotoxins on their own, yet it has been demonstrated that the two relatively unreactive radicals can rapidly combine (k=3.7xc3x97107 Mxe2x88x921 sxe2x88x921) under physiological conditions to the strong oxidizing agent peroxynitrite (ONOOxe2x88x92). This reaction is about 3 times faster than the detoxifying catabolism of superoxide by superoxide dismutase (SOD). It is believed that the formation of peroxynitrite is an important factor in the oxidative damage associated with ischemia/reperfusion. A variety of pathologies are associated with the formation of peroxynitrite. Peroxynitrite is invariably formed in larger amounts when more NO is produced, and/or when an elevated level of superoxide prevails. In this regard, pathologies such as diabetes, atherosclerosis, and ischemia-reperfusion injury, are associated with oxidative stress characterized by an elevated level of superoxide that can lead to increased peroxynitrite formation. Also when glutathione detoxification mechanism against peroxynitrite is impaired critical concentrations of peroxinitrite may occur. Recent evidence also suggests multiple sclerosis and Alzheimer""s disease are associated with peroxynitrite formation. In addition, peroxynitrite has also been implicated during sepsis and adult respiratory distress syndrome. Ischemia and reperfusion are accompanied by an increase in superoxide due to the activation of xanthine oxidase and NAPDH oxidase, respectively. Thus, peroxynitrite is likely to be implicated in a number of pathologies in which an imbalance of NO and superoxide occurs.
Several factors can contribute to reduced bioavailability of NO, ranging from impaired production to increased degradation, depending on the risk factors involved. NO is synthesized by dimers of the 130 kD enzyme endothelial NO synthase in a reaction where arginine is oxidized to NO and citrulline. It has been shown that eNOS produces superoxide radicals as well as NO. Under physiological conditions, NOS predominantly produces NO, controlled by the regulatory co-enzyme calmodulin, the substrate arginine and the cofactor tetrahydrobiopterin (BH4). Under pathophysiological conditions, such as dyslipidemia, production shifts from NO to superoxide. Clinical studies have shown impaired NO bio-availability in patients with (risk factors for) atherosclerosis. Evidence has accumulated showing that increased production of superoxide and increased degradation of NO by superoxide, rather than impaired formation of NO is the predominant cause of impaired NO bioavailability in early atherosclerosis. These observations indicate that atherogenesis is linked to a pathological imbalance between NO and superoxide, rather than reduced NO production per se.
The level of superoxide can be lowered by substances showing a relevant scavenging capacity for superoxide radicals. Measurements revealed that arginine does not react with superoxide. However, both arginine and tetrahydrobiopterin (BH4) are required to minimize or abolish superoxide formation by NOS. Tetrahydrobiopterin (BH4) shows a reaction rate with superoxide which is roughly 2 fold smaller than that of the potent antioxidant ascorbic acid and for folic acid, folates or derivatives thereof (as an example 5-methyl-(6 S)- and (6R)-tetrahydrofolic acid have been measured), the reaction rates are about 20 times slower than that of ascorbic acid. Beside of its tenfold lower scavenging capacity folic acid, folates or derivatives thereof are different from tetrahydrobiopterin (BH4) or derivatives thereof in that achievable plasma concentrations are far lower. Upon standard oral suppletion of folic acid (5 mg p.o.) systemic plasma concentrations of 5-methyltetrahydrofolic acid up to ca. 150 nM are achieved whereas upon intra-arterial infusion values of 250 nM were reached. Both these interventions have been shown to result in an improvement in NO-availability in hypercholesterolemic patients. Still these levels of folic acid, folates or derivatives thereof remain orders of magnitude below those of ascorbic acid (concentrations up to 50 xcexcM).
Despite of the situation that it has been known that xe2x80x9ca scavenging effect of BH4 had been remarkedxe2x80x9d [Vasquez-Vivar, J. et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 9220-9225], xe2x80x9cexogenous BH4 is capable of restoring impaired NO activity in prehypertensive ratsxe2x80x9d [Cosentino, F. et al., J. Clin. Invest., 1998, 101, 1530-1537], xe2x80x9cexogenous BH4 is capable of restoring impaired NO activity in hypercholesterolemia patientsxe2x80x9d [Stroes, E. at al., J. Clin. Invest., 1997, 99, 41-46], xe2x80x9cexogenous BH4 is capable of restoring impaired NO activity in diabetic patientsxe2x80x9d [Pieper, G. M., J. Cardiovasc. Pharmacol., 1997, 29, 8-15], xe2x80x9cfolate therapy improves NO activity during hypercholesterolemia in vivouxe2x80x9d [Woo, K. S. at al., Circulation, 1998, 97, I-165-166] and [Verhaar, M. C. et al., Circulation, 1998: 97 (3), 237-241], xe2x80x9cfolic acid and its active form 5-MTHF restore impaired NO bioavailability in dyslipidemic conditionsxe2x80x9d [Wilmink, H. et al., Arteriosclerosis Thromb Vasc Biol, 2000; 20 (1), 185-8] and [Verhaar, M. C. et al., Circulation, 1998; 97 (3), 237-241], xe2x80x9cclinical studies have revealed that the impairment of endogenous vasodilator function observed with hypercholesterolemia is reversible by administration of L-argininexe2x80x9d [Creager, M. A. et al., Clin Invest. 1992, 90, 1248-1253] and xe2x80x9cFolic acid supplementation improves arterial endothelial function in adults with realtive hyperhomocysteinemiaxe2x80x9d [Woo, K. S. et al., J. Am. College of Cardiology, 1999, 34 (7), 2002-2006] the use of at least folic acid or a folate and tetrahydrobiopterin (BH4) or derivatives thereof together with pharmaceutically compatible active and adjuvant substances, such as arginine for the production of a pharmaceutical preparation suitable for influencing the nitric oxide (NO) level has neither been proposed nor described hitherto.
This is probably due to the situation that it has been postulated that xe2x80x9cMTHF had no direct effect on in vitro NO production by eNOSxe2x80x9d [Verhear, M. C. et al., Circulation, 1998; 97 (3), 237-241].
It has been found that the use of pharmaceutical preparations containing at least folic acid or a folate and tetrahydrobiopterin (BH4) or derivatives thereof influences the nitric oxide (NO) level, and in particular affects the enzymatic activity of nitric oxide synthase (NOS) by reducing superoxide production and enhancing nitric oxide (NO) synthesis. This effect occurs in absence of negative changes in other risk factors, e.g. lipids, blood pressure and homocysteine.
Especially surprising is this effect as in pterin-free eNOS folic acid, a folate or a derivative thereof does not affect the enzymatic activity of nitric oxide synthase (NOS), neither with regard to NO, nor to superoxide production, whereas in partially pterin-repleted eNOS folic acid, a folate or a derivative thereof have the claimed strong effect on the activity of the enzyme; i.e. they enhance NO production concomitant with a decreased production of superoxide. The beneficial vascular effect of folic acid or a folate together with at least tetrahydrobiopterin (BH4 ) or derivatives thereof cannot be attributed solely to direct scavenging of superoxide.
Folic acid, a folate or a derivative thereof refers to folic acid (pteroylmonoglutamate), one or more of the folylpolyglutamates, compounds in which the pyrazine ring of the pterin moiety of folic acid or of the folylpolyglutamates is reduced to give dihydrofolates or tetrahydrofolates, or derivatives of all the preceding compounds in which the N-5 or N-10 positions carry one carbon units at various levels of oxidation, or pharmaceutically compatible salt thereof or a combination of two or more thereof. Especially means folic acid, a folate or a derivative thereof folic acid, dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, 5,10-methenyltetrahydrofolate, 5,10 -formiminotetrahydrofolate, 5-formyltetrahydrofolate (leucovorin), 10-formyltetrahydrofolate 10-methyltetrahydrofolate, pharmaceutically compatible salts thereof, or a combination of two or more thereof.
Reduced folates can be converted into one another according to the well known folate metabolism. 5-methyltetrahydrofolic acid and the pharmaceutically compatible salts thereof are preferably used, however, since 5-methyltetrahydrofolic acid is directly involved together with tetrahydrobiopterin in such functions as the biosynthesis of dopamine, norepinaphrine and serotinine by the hydroxylation of phenylalanine and the regeneration of BH4 by the reduction of the quinonoid 7,8-dihydrobiopterin to tetrahydrobiopterin. This applies in particular when there is an existing methylenetetrahydrofolate reductase deficiency, wherein this deficiency implies disorders such as restricted functionality or lack of activity, for example. The existence of thermolabile methylenetetrahydrofolate reductase should be mentioned here as the most frequent example of a methylenetetrahydrofolate reductase deficiency. Under these circumstances, especially 5-methyltetrahydrofolic acid is only available in a limited amount.
Within all folates or a derivatives thereof both the natural and the unnatural diastereoisomers, pharmaceutically compatible salts thereof and any mixtures of the isomers and the salts, but especially the natural diastersoisomeric forms such as 5-methyl-(6 S)-tetrahydrofolic acid are applicable.
Tetrahydrobiopterin (BH4) refers to all the natural and the unnatural forms of tetrahydrobiopterin, pharmaceutically compatible salts thereof and any mixtures of the isomers and the salts, but especially the natural diastereoisomeric form (6R)-L-erythro-tetrahydrobiopterin is applicable.
Arginine refers to the both the natural and unnatural isomeric form of arginine, pharmaceutically compatible salts thereof and any mixtures of the isomers and the salts, but especially the natural isomeric form L-arginine is applicable.